Novel Thiazolidinone-Azole Hybrids: Design, Synthesis and Antimycobacterial Activity Studies.

To develop novel antimycobacterial agents, a new series of thiazolidinone-azole hybrids 4a-b, 5a-b and 6-13 were designed and synthesized. Thiazolidin-4-ones (4a-b and 5a-b) were obtained by the reaction of Schiff bases and hydrazones (2a-b and 3a-b) with mercaptoacetic acid. 5-Benzylidene derivatives (6-13) were gained by treatment of 5a-b with appropriate benzaldehydes according to Knoevenagel condensation. To evaluate their structures 1H NMR, IR, mass spectrometry and elemental analysis data were used. The target compounds were screened for their antimycobacterial activity against M. tuberculosis H37Rv strain using the microplate alamar blue assay method. Among them, 6, 10 and 12 (MIC: 14.27-14.74 μM) were found as most active compounds in the series. It was seen that both phenylamino and benzylidene substitutions on thiazolidin-4-one ring caused an improvement in the antimycobacterial activity.


Introduction
Tuberculosis (TB) is one of the most common infectious diseases known to man. According to the WHO report in 2012, there were almost 9 million new cases of tuberculosis and 1.4 million tuberculosis deaths (1). The problems with current TB treatment are complex and include: a prolonged standard course regimen of six months, which often result in patient noncompliance; emergency of extremely drug-resistant tuberculosis (XDR-TB) strains; lack of effective drugs against the latent state (2-4). Therefore, there is an urgent need for antitubercular agents with improved properties such as enhanced activity against MDR strains, reduced toxicity, shortened duration of treatment.
These facts have led us to study on thiazolidinone-azole hybrid compounds which are expected to show antitubercular activity.

Chemistry
Melting points were determined with a Thomas-Hoover Capillary Melting Point Apparatus (Philadelphia, PA, USA) and are uncorrected. ATR-FTIR spectra were obtained using the MIRacle ATR accessory (Pike technologies) in conjunction with a Spectrum BX FTIR spectrometer (Perkin Elmer) and were reported in cm -1 . The 1 H NMR (400 MHz) spectra (DMSO-d 6 ) were recorded on a Varian Mercury 400 FT NMR spectrophotometer (Varian Inc, Palo Alto, CA, USA) using TMS as an internal reference (Chemical shift represented in δ ppm).The ESI-MS spectra were measured on a micromass ZQ-4000 single quadruple mass spectrometer. Elemental analyses (C, H, N) were performed on Leco CHNS 932 analyzer (USA).

General procedure for the preparation of Schiff bases 2a-b and hydrazones 3a-b
Equimolar amounts of an appropriate benzaldehyde and anilin/phenylhydrazine were refluxed in methanol in presence of acetic acid (1 drop) as a catalytic reagent for 4 h. The solvent was evaporated and the crude mixture was used without any purification in the next step.

Antimycobacterial Activity Assay
The target compounds were tested for their antimycobacterial activity in vitro against M. tuberculosis H 37 R v using the microplate alamar blue assay (MABA) method (27) in duplicate. Isoniazid, rifampin, ethambutol and ciprofloxacin were used as positive and DMSO as negative control. Compound stock solutions were prepared in DMSO. Sterile deionized water (200 μL) was added to all outer-perimeter wells of sterile 96-well plates to minimize evaporation of the medium in the test wells during incubation. The wells received 100 μL of Middlebrook 7H9GC broth and two fold serial dilutions of the target compounds/positive controls were prepared in a volume of 100 μL directly on the plate. 100 μL of MTB inoculum was added to the wells. The plates were sealed with parafilm and were incubated at 37 °C for five days. 50 μL of a freshly prepared 1:1 mixture of Alamar Blue (Accumed International, Westlake, Ohio) reagent and 10% Tween 80 was added to the plates and incubated at 37 °C for 24 h. A blue colour in the well was interpreted as no growth, and a pink colour was scored as growth. The MIC was determined as the lowest drug concentration which prevented a colour change from blue to pink. MICs of the compounds were reported in Table 1.

Conflict of Interest
We declare that we have no conflict of interest with respect to this study.
In the IR spectra of compound 4a-b and 5ab, the presence of the C=O stretching bands at around 1680 cm -1 indicated the thiazolidin-4-one ring closure. In the 1 H NMR spectra of these compounds, the signals of nonequivalence methylenic protons of the thiazolidin-4-one ring were observed as two doublets at around 4.0 ppm (H A ) and as doublet at around 3.9 ppm (H B ) because of geminal (J AB of 15.6 Hz) and long range coupling (~J Ax of 1.6 Hz) with C 2 proton. Furthermore, the signal of the C 2 proton of the thiazolidin-4-one ring was seen as singlet or doublet at around 6.0 ppm. In the 1 H NMR spectra of 6-13, disappearance of the signals of methylenic protons and formation of a new signal at around 7.5 ppm proved benzylidene substitution to the thiazolidin-4-one ring. In the mass spectra, all compounds displayed molecular ion peaks which confirmed their molecular weight.

Antimycobacterial Activity
The target compounds (4a-b, 5a-b and 6-13) were evaluated for their antimycobacterial activity in-vitro against Mycobacterium tuberculosis H 37 R v using the microplate alamar blue assay method (27)  These results led us to focus on 3 -( p h e n y l a m i n o ) -1 , 3 -t h i a z o l i d i n -4ones. As a continuation of our study, we condensed several benzaldehydes to obtain 5-(substitutedbenzylidene)-3-(phenylamino)-1,3-thiazolidin-4-ones (6-13). When we compared the activity results of 5a and 5b with 6-9 and 10-13 respectively, it was observed that benzylidene substitution to the 3-(phenylamino)-1,3-thiazolidin-4-ones enhanced the activity (except 11). However 12 (MIC: 14.27 μM) was the most active derivative in this series, introducing the substituents at the 4 th position of the benzylidene ring decreased the activity in generally. According to the activity results of 6 and 10 (with MIC of 14.70 and 14.74 μM respectively), it can be assumed that non-substitutedbenzylidene structure is a favorable moiety for the activity. Comparing the compounds bearing triazole moiety (6-9) with that of imidazole analogues (10-13), it was seen that the type of the azole ring did not cause any remarkable difference on the activity except methyl substituted derivatives (8 with MIC of 56.94 μM and 12 with MIC of 14.74 μM). Furthermore, none of the compounds in the series were found as active as standard compounds (Table 1).
In summary, a series of novel thiazolidin-4-one derivatives 4a-b, 5a-b and 6-13 were designed, synthesized to evaluate their antimycobacterial activity. Among the target compounds, 6, 10 and 12 were found as most active compounds in the series. It was seen that both phenylamino and benzylidene substitutions on thiazolidin-4-one ring caused an improvement in the antimycobacterial activity.